The major objective of this research is to learn the detailed mechanisms by which adenylate kinase, pyruvate kinase and Staphylococcal nuclease catalyze nucleophilic substitutions at phosphorus, accelerating the rates of these reactions by huge factors approaching 1016-fold. With pyruvate kinase, we also wish to learn how the enzyme enolizes pyruvate prior to its phosphorylation by MgATP, and how the rates of these processes are positively controlled by fructose diphosphate in the allosteric enzymes from yeast and liver, and negatively controlled by covalent phosphorylation in the hepatic enzyme. Also under investigation are the mechanistically related phosphoryl transfer reactions catalyzed by creatine kinase and cAMP-dependent protein kinase, and the enolization reactions catalyzed by delta 5-3-ketosteroid isomerase, glyoxalase I, and yeast aldolase. High resolution nuclear magnetic resonance method using paramagnetic probes, nuclear Overhauser effects, and computerized conformational search procedures, as well as pulsed EPR and X-ray absorption spectroscopy are used to elucidate the ligands of essential metal cofactors, the conformations, arrangements and exchange rates of enzyme-bound substrates, and the nature of the functional groups on the enzyme(s) which interact with the substrates. Such information also permits us to position or dock substrates into the X-ray structures of enzymes. The solution structure and interactions of small peptide fragments of adenylate kinase and creatine kinase, 45 to 88 residues in length, respectively, which retain the ability to bind MgATP tightly and, in the case of the adenylate kinase peptide 1-45, hold MgATP in a conformation similar to that bound on the complete enzyme, are being studied by 1- and 2-dimensional NMR methods. NMR studies of such "isolated active sites" provide detailed information on potential catalytic residues, which will be further tested by the substrate binding properties of synthetically altered peptides and by kinetic, metal, and substrate binding studies of the complete enzymes in which these residues have been mutated. Our site- specific mutations of individual catalytic residues have provided qualitative support for our proposed mechanisms of Staphylococcal nuclease and of ketosteroid isomerase. It is important to determine whether the quantitative effects on catalysis of mutating individual residues may be multiplied together to explain the large overall rate accelerations produced by these enzymes. If so, then a quantitative understanding of enzyme catalysis is at hand. We propose to examine this fundamental point by studying the kinetic, substrate binding, and structural effects of double mutants involving pairs of catalytic residues on Staphylococcal nuclease and ketosteroid isomerase. The purpose of studying several enzymes which catalyze chemical reactions of the same class is to elucidate general principles of enzyme chemistry, and to develop and critically compare various spectroscopic approaches to enzyme structure and mechanism.